Activity-Dependent Regulation Of Oligodendrocyte Function and Myelination by Misti Lane Malone
Dendropanax morbiferus leaf extract facilitates oligodendrocyte … · 2019-09-20 · For plant...
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royalsocietypublishing.org/journal/rsos
ResearchCite this article: Kim J-Y, Yoon J-Y, Sugiura Y,
Lee S-K, Park J-D, Song G-J, Yang H-J. 2019
Dendropanax morbiferus leaf extract facilitates
oligodendrocyte development. R. Soc. open sci. 6:
190266.
http://dx.doi.org/10.1098/rsos.190266
Received: 13 February 2019
Accepted: 4 June 2019
Subject Category:Cellular and molecular biology
Subject Areas:molecular biology/cellular biology/neuroscience
Keywords:oligodendrocyte, differentiation, myelin,
multiple sclerosis, Dendropanax morbiferus
Author for correspondence:Hyun-Jeong Yang
e-mail: [email protected]
& 2019 The Authors. Published by the Royal Society under the terms of the CreativeCommons Attribution License http://creativecommons.org/licenses/by/4.0/, which permitsunrestricted use, provided the original author and source are credited.
Electronic supplementary material is available
online at https://dx.doi.org/10.6084/m9.figshare.
c.4544111.
Dendropanax morbiferusleaf extract facilitatesoligodendrocyte developmentJi-Young Kim1, Ju-Young Yoon2, Yuki Sugiura3,
Soo-Kyoung Lee4, Jae-Don Park5, Gyun-Jee Song6
and Hyun-Jeong Yang2,7
1Department of Anesthesiology and Pain Medicine, Yonsei University College of Medicine,Seoul 120-749, Republic of Korea2Department of Integrative Biosciences, University of Brain Education, Cheonan 31228,Republic of Korea3Department of Biochemistry and Integrative Medical Biology, School of Medicine,Keio University, Tokyo 160-8582, Japan4Department of Health Science and Daily Sports, Global Cyber University, Cheonan 31228,Republic of Korea5Cheju Halla University, Jeju 63092, Republic of Korea6Department of Medical Science, International St Mary’s Hospital, Catholic KwandongUniversity, Gangneung, Republic of Korea7Korea Institute of Brain Science, Seoul, Republic of Korea
H-JY, 0000-0002-5262-6721
Treatment of multiple sclerosis is effective when anti-
inflammatory, neuroprotective and regenerative strategies
are combined. Dendropanax morbiferus (DM) has anti-
inflammatory, anti-oxidative properties, which may be
beneficial for multiple sclerosis. However, there have been no
reports on the effects of DM on myelination, which is critical
for regenerative processes. To know whether DM benefits
myelination, we checked differentiation and myelination of
oligodendrocytes (OLs) in various primary culture systems
treated with DM leaf EtOH extracts or control. DM extracts
increased the OL membrane size in the mixed glial and pure
OL precursor cell (OPC) cultures and changed OL-lineage
gene expression patterns in the OPC cultures. Western blot
analysis of DM-treated OPC cultures showed upregulation
of MBP and phosphorylation of ERK1/2. In myelinating
cocultures, DM extracts enhanced OL differentiation,
followed by increased axonal contacts and myelin gene
upregulations such as Myrf, CNP and PLP. Phytochemical
analysis by LC-MS/MS identified multiple components from
DM extracts, containing bioactive molecules such as
quercetin, cannabidiol, etc. Our results suggest DM extracts
enhance OL differentiation, followed by an increase in
membrane size and axonal contacts, thereby indicating
enhanced myelination. In addition, we found that DM
roya2
extracts contain multiple bioactive components, warranting further studies in relation to findingeffective components for enhancing myelination.
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1. IntroductionDendropanax morbiferus (DM) grows in eastern Asia, central and south America and the Malaysian
peninsula [1]. Recent scientific researches on DM have shown its antioxidant effects [2,3], protective
effects on human dopaminergic cells [4], induction of human leukaemia U937 cell apoptosis [5] and
of human osteosarcoma cell autophagy [6], anti-thrombotic effects [7] and anti-inflammatory [8],
particularly neuroinflammatory, effects [9].
Multiple sclerosis is a major demyelinating disease of the central nervous system leading to focal
plaque of primary demyelination and diffuse neurodegeneration in the grey and white matter of the
brain and spinal cord [10].
Patients with multiple sclerosis show increased oxidative stress markers and inflammation [11].
Recent studies on DM suggest that DM might be effective on multiple sclerosis symptoms because of
its antioxidant [2,3], cell-protective [4] and anti-inflammatory properties [8,9].
Remyelination fails in many chronically demyelinated multiple sclerosis plaques [12,13]. The
remyelination failure is mainly attributable to reduced oligodendrocyte precursor cell (OPC)
recruitment and differentiation [14]. Among these events, it is thought that disrupted differentiation is
the major time-limiting factor because OPC mitogen and recruitment factor PDGF overexpression do
not increase remyelination [15]. Currently, it is understood that oligodendrocyte (OL) differentiation
failure is the biggest cause of remyelination failure [16]. Study of various developmental markers of
OLs has shown that OPC is not fully differentiated in multiple sclerosis lesions [12,13,17,18].
Therefore, enhancement of OL differentiation and myelination is an important problem to solve in
relation to multiple sclerosis treatment.
Although recent research has reported that DM has anti-oxidative, cell-protective and anti-
inflammatory properties, which can be useful in multiple sclerosis treatment, the link between DM’s
benefits and myelin-forming OLs has not been investigated yet. In the present study, we aimed to
reveal the effects of DM on OL differentiation, and myelination using multiple primary culture
systems, including in vitro myelination cultures.
2. Material and Methods2.1. MiceTimed pregnant females (day 13.5 of pregnancy) or pups (postnatal day 0–2) of CrljOri:CD1(ICR) mouse
line were purchased from ORIENT BIO Inc. (Seongnam, Korea). All experiments were performed in
compliance with the relevant laws and institutional guidelines and were approved by the University
of Brain Education’s Animal Care and Use Committee (approval no. 2017-AE-01).
2.2. Preparation of plant extractsThe leaves, stem barks and roots of 6-, 20- and 50-year-old DM were collected on Jeju island and were
identified and provided by Mago-Yeongnong-Johap (Jeju, Korea). Other dried plants, except DM,
were identified and purchased from Jirisan Cheongjung Yakcho (Sancheong, Kyoungnam, Korea):
Eucommia ulmoides (EU), Achyranthes aspera (AcA), Xanthium strumarium (XS), Astragalus membranaceus(AM), Artemisia princeps (ArP), Actinidia polygama (AP), Rehmannia glutinosa (RG), Ledebouriella seseloides(LS) and Artemisia annua (AA). The product year of these plants was 2016. All the specimens were
deposited in the University of Brain Education Herbarium. The deposition numbers are as follows:
6-year-old DM stem (2017-05-3), leaf (2017-12-1) and root (2017-12-2); 20-year-old DM leaf (2017-13);
50-year-old DM stem (2017-05-2) and leaf (2017-14); EU (2017-03); AcA (2017-07); XS (2017-08); AM(2017-09); ArP (2017-04); AP (2017-01); RG (2017-06); LS (2017-11) and AA (2017-10). Dried plant
samples were chopped and ground into a fine powder because lowering particle size increases
surface-contact area between samples and extraction solvents, resulting in efficient extraction [19].
Eight grams of each powder were soaked into EtOH with up to 50 ml volume. Plant samples were
royalsoc3
incubated in the shaking rotator for one week at room temperature. After one week, the insolublematerials were removed by centrifugation and the resulting supernatant was collected and
concentrated by evaporation at 708C. The extracts were adjusted to 8 g ml21 and stored in aliquots at
2208C [19,20].
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2.3. Cell culturesMixed glial cultures were prepared from cortices of mice at postnatal days 0–2 on poly-D-lysine
(PDL)-coated flasks or coverslips and were maintained in DMEM/F-12 medium containing 10% fetal
bovine serum, 5% horse serum and 1� penicillin–streptomycin (Gibco). For OPC pure cultures, OPCs
were isolated by shaking from the flasks containing mixed glial cultures on days in vitro (DIV) 10.
After removing the astrocytes by dish-panning, the OPCs were seeded on PDL-coated coverslips and
were maintained in Sato medium (DMEM containing 1� B-27 supplement, 1� Glutamax, 1�penicillin–streptomycin, 1% horse serum, 1� sodium pyruvate, 0.34 mg ml21 T3 and 0.4 mg ml21 T4).
For cocultures, mouse dorsal root ganglia (DRG) neuronal cultures and mouse glial mixed cultures
were separately prepared in advance. DRG neurons were prepared as described previously [21]. The
OPCs were isolated by shaking and were seeded on DRG neuronal cultures and were maintained
in coculture medium (DMEM containing B-27 supplement, N-2 supplement, 5 mg ml21 N-Acetyl-
cysteine, 5 mM forskolin and penicillin–streptomycin). For plant extract treatment, the above prepared
plant extracts were diluted with the medium into 1 : 1000 (high, if not indicated) or 1 : 100 000 (low)
and filtered through a 0.22 mm syringe filter. Cells were incubated with the plant extract-diluting
medium or control medium for DIV 8–12 in the mixed glial cultures, for DIV 1–3 in the pure OPC
cultures and for DIV 1–7 or 1–12 for DRG/OPC cocultures. Control or plant extract-containing
medium was changed with fresh medium every two days. If not indicated, the leaf part of six-year-
old DM was used in the treatment.
2.4. ImmunofluorescenceFor immunocytochemistry, the cells were fixed with 4% paraformaldehyde (PFA)/phosphate-buffered
saline (PBS) for 15 min at room temperature; washed with PBS; and blocked for 45 min at room
temperature with blocking solution (PBS containing 5% normal donkey serum, 0.5% Triton X-100 and
0.05% sodium azide). The samples were incubated overnight at 48C with primary antibodies diluted
in blocking solution, washed three times in PBS, incubated for 45 min with secondary antibodies and
washed in PBS and mounted with mounting medium (Vectashield H-1400, vectorlabs). For primary
antibodies, the following antibodies were used: rat monoclonal antibody to MBP (1 : 300, MAB386,
Chemicon), mouse hybridoma supernatants to O4 (1 : 5), rabbit polyclonal antibodies to Olig2 (1 : 500,
AB9610, Chemicon) and Caspr (1 : 1000) [22]. Secondary antibodies were obtained from Jackson
Immunoresearch. DAPI was obtained from Invitrogen.
2.5. Real-time PCRTotal RNA was isolated with TRIzol (Sigma) from cultured OPC cells on DIV 3 or myelinating cultures
on DIV12. The isolated RNA was treated with DNaseI to eliminate genomic DNA before reverse
transcription. Mouse cDNAs were prepared using SuperScript II Reverse Transcriptase (Invitrogen).
Specific primer sets were used for Olig1 (forward, 50-GCTCGCCCAGGTGTTTTGT-30; reverse,
50-GCATGGAACGTGGTTGGAAT-30), Id2 (forward, 50-CCTGCATCACCAGAGACCTG-30; reverse,
50-TTCGACATAAGCTCAGAAGGGAA-30), Ascl1 (forward, 50-CAACCGGGTCAAGTTGGTCA-30;
reverse, 50-CTCATCTTCTTGTTGGCCGC-30), MBP (forward, 50-CCAGAGCGGCTGTCTCTTCC-30;
reverse, 50-CATCCTTGACTCCATCGGGCGC-30), Myrf (forward, 50-TGGCAACTTCACCTACCACA-30;
reverse, 50-GTGGAACCTCTGCAAAAAGC-30), CNP (forward, 50-GTTCTGAGACCCTCCGAAAA-30;
reverse, 50-CCTTGGGTTCATCTCCAGAA-30), PLP (forward, 50-GGTACAGAAAAGCTAATTGAGA
CC-30; reverse, 50-GATGACATACTGGAAAGCATGA-30) and GAPDH (forward, 50-GGTCGGTGTGAA
CGGATTTG-30; reverse, 50-TCGTTGATGGCAACAATCTCCACT-30). Real-time PCR was performed in
the Step One Plus Real-Time PCR System (Applied Biosystems) using SYBR Green mix. All reactions
were performed in triplicate and GAPDH was used for normalization.
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2.6. Western blot analysisWestern blot analysis was performed using OPC cultures lysed in loading buffer, and
chemiluminescence was detected using Amersham Imager 600 (GE Healthcare Life Sciences). Rat
antibody against MBP (1 : 500, MAB386, Chemicon), rabbit antibodies against phospho-p42/44 (1 :
1000, 4370P, Cell signaling), and b-actin (1 : 5000, Abcam) were used for immunoblotting.
2.7. Image analysisImages were obtained using a Leica TCS SPE microscope and Axio Imager Z1 equipped with Apotome
(Carl Zeiss). Image analysis was performed using Image J. The images were given to the investigators
without the sample names but only with numbers for analysis. Thus, the images were scored blinded
to the plant names. For MBPþ or Casprþ area per cell, the images were manually analysed in a random
manner to detect single cells under the same threshold throughout all images. If distinguishing a single
cell was difficult because of the density, the cells were excluded from the analysis. For Olig2þ or Dapiþ
cell number, the cells were automatically detected and counted under the same threshold throughout
all images in Image J. For O4þ or MBPþ area per field, marker-positive areas were automatically
detected and measured under the same threshold throughout all images in Image J.
2.8. Statistical analysesAll graph data are presented as the mean+ s.e.m. Statistical analyses were performed using unpaired
Student’s t-test with two tails, unequal variance. Sample size was based on similar studies in the field.
2.9. Non-targeted metabolome analysisDM leaf ethanol extracts were stored at 2808C until analyses and phytochemicals were analysed by liquid
chromatography (LC)-mass spectrometry (MS)/MS. For non-targeted analysis, MS (Q-Exactive focus,
Thermo Fisher Scientific, San Jose, CA, USA), which enables us to perform highly selective and
sensitive metabolite quantification owing to the Fourier transfer MS principle, was connected to a high-
performance liquid chromatography (Ultimate3000 system, Thermo Fisher Scientific). The samples were
resolved on Acculaim C18 (2.1 mm ID � 150 mm, 3 mm particle, Thermo Fisher Scientific), using a step
gradient with mobile phase A (60 : 40 (v/v) of water: acetonitrile in 10 mM ammonium formate and
0.1% formic acid) and mobile phase B (90 : 10 (v/v) of isopropyl alcohol: acetonitrile in 10 mM
ammonium formate and 0.1% formic acid) at ratios of 68 : 32 (0–4 min), 55 : 45 (4–5 min), 48 : 52
(5–8 min), 34 : 66 (8–11 min), 30 : 70 (11–14 min) and 25 : 75 (14–18 min), at a flow rate of 0.2 ml min21
and a column temperature of 458C. The Q-Exactive focus mass spectrometer was operated under an
ESI-positive mode for all detections. Full mass scan (m/z 50–900), followed by three rapid data-
dependent MS/MS, was operated at a resolution of 70 000. The automatic gain control target was set at
3 � 106 ions, and the maximum ion injection time was 100 ms. Source ionization parameters were
optimized with the spray voltage at 3 kV and the other parameters were as follows: transfer temperature
at 3208C, S-Lens level at 50, heater temperature at 3008C, sheath gas at 36 and auxilliary gas at 10.
Compound Discoverer 2.0 (Thermo Fisher Scientific) was used for the non-targeted metabolomics
workflow as described in Zhou [23]. Briefly, this software first aligned the total ion chromatograms of
different samples along the retention time. Then the detected features were extracted and merged into
components. The resulting compounds were identified by both (i) formula prediction based on
accurate m/z value and isotope peak patterns, and (ii) MS/MS structural validation querying to m/zcloud database (https://www.mzcloud.org/). Moreover, formula predicted signals were assigned into
candidate compounds by database search (ChemSpider database; http://www.chemspider.com/).
3. Results3.1. Dendropanax morbifera extracts increased the oligodendrocyte membrane size in the
mixed glial culturesTo check whether DM extract conferred any effects on the formation of the OL membrane sheath, we
performed mixed glial cultures from the cortices of the postnatal days 0–2 ICR pups on PDL-coated
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coverslips. The cultures were initially incubated in the regular glial medium, and after eight days themedium was changed to Sato medium containing either the ethanol control (figure 1a,c) or
the following medicinal plant extracts, which have been appeared as their neurological recovery
function in traditional literature [24]: DM (figure 1b,d ), EU, AcA, XS, AM, ArP, AP, RG, LS and AA(figure 1a–e; electronic supplementary material, figure S1). On DIV 12, the cultures were fixed and
stained with MBP to determine the size of the OL membrane sheath. MBP is the major marker of
mature myelinating OLs [21]; therefore, we used MBP-positive percentage area per field on DIV 12 as
the indication of membrane formation. Using Image J, MBP-positive percentage area per field was
measured. The pictures were taken in low magnification to include as many mature OLs as possible.
Many of the investigated medicinal plant extracts in the study including DM leaf extract induced
increases in MBP-positive membrane size compared with EtOH control (figure 1e). ArP and DMshowed the highest effects on MBP-positive percentage area per field in the glial mixed culture
system. The glial mixed cultures contain three major glial cell types: OLs, astrocytes and microglia.
Therefore, the effects of DM on OL membrane sheath formation may derive not only from the OLs
but also from the combined effects of the three cell types, because astrocytes and microglia also affect
myelin synthesis [25,26].
3.2. Dendropanax morbiferus extracts increased the size of oligodendrocyte membrane sheathin the isolated oligodendrocyte precursor cell cultures
To clarify whether the changes in membrane size by DM extracts derive directly from OL or indirectly
through astrocytes or microglia, we isolated OPCs from the mixed glial culture on DIV 10 by shaking.
Isolated OPCs were seeded on PDL-coated coverslips and incubated in Sato medium containing
differentiating hormones T3 and T4 [21]. On DIV 1, the medium was changed to fresh medium
containing either the control EtOH (figure 1f,l ) or the following plant extracts: DM (figure 1g,m), AA(figure 1h), AP (figure 1i), EU (figure 1j ), LS (figure 1k), DF and ArP (figure 1n). The cultures were
fixed on DIV 4 and were then used for MBP staining. The images were taken and blindly analysed
using Image J. MBP staining images were converted into 8 bit pictures (electronic supplementary
material, figure S2 h), and all single cells per field were identified manually and numbered (electronic
supplementary material, figure S2i). MBP-positive area per cell was calculated using the Image J
(figure 1n). In pure OPC cultures, DM extract-treated OLs showed the largest membrane size
(figure 1n), suggesting that the DM extracts contain components that directly enhance OL membrane
synthesis independent of other glial cell types. Unlike the results in the glia mixed culture, ArPshowed only half of the membrane size compared with DM-treated OLs in pure OPC cultures,
suggesting its action may require the combinatory mechanism with astrocytes or microglia.
3.3. Dendropanax morbiferus showed different effects on oligodendrocyte membrane synthesisdepending on its part
DM may have different biological activities depending on its parts [27,28]. To clarify the part of DM that
would be the most effective for OL membrane synthesis, we tested leaves, stems and roots of DM. Pure
OPC cultures were prepared and control EtOH or DM extracts were added from DIV 1, and the cultures
were fixed on DIV 3. Compared with control EtOH, DM leaf extracts induced a marked increase in MBP-
positive area per cell (***p , 0.001, n ¼ 3 experiments, Student’s t-test), DM roots showed a relatively
small increase (*p , 0.05, n ¼ 3 experiments, Student’s t-test), and DM stem did not show any
significant changes (figure 1o). MBP-positive areas per cell were dependent on the concentration of the
extracts (electronic supplementary material, figure S2j). A higher concentration of DM leaf led to a
larger OL membrane size than lower concentration. This suggests that the effective components of
DM on OL membrane synthesis exist strongly in the leaves of DM.
3.4. Dendropanax morbiferus leaf extracts induced changes in gene expressions relatedwith OL differentiation
To check whether DM leaf altered the gene expression in OLs, we used pure OPC cultures and treated
them with control EtOH or DM leaf extract of low or high concentrations (figure 2a–d, see Material and
Methods for concentrations) from DIV 1 and collected cells to isolate RNA at DIV 3, followed by cDNA
glia mixed culture OPC culture OPC culture
EtOH
EtOH EtOH
Dendropanax m. Dendropanax m.
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Figure 1. Dendropanax morbiferus leaf EtOH extract treatment increased the size of OL membrane sheath in the mixed glial cultureas well as pure OL culture. (a – e) Primary mixed glial cultures were treated with the medium containing EtOH or several medicinalplant EtOH extracts during DIV 8 – 12 and fixed at DIV 12 for MBP staining to analyse the membrane size. Compared with EtOH-treatedcultures (a,c), the size of OL membrane sheath was increased by the treatment of medicinal plant extracts including DM (b,d; electronicsupplementary material, figure S1). (e) Total summary of MBP-positive percentage area per field depending on the treatment. Amongthe investigated medicinal plants, ArP and DM induced the highest MBP-positive percentage area per field in the glia mixed culture.( f – n) OPCs were isolated from the mixed glial cultures on DIV 10 and seeded on PDL-coated coverslip. On DIV 1, the OPCs were treatedwith the EtOH- or the indicated medicinal plant extract-containing medium and fixed on DIV 4 for immunostaining with MBP antibody.Dendropanax morbiferus-treated OPCs showed the highest MBP-positive area per cell, while ArP-treated OPCs indicated the half of themembrane size of DM-treated OPCs. (o) MBP-positive area per cell depending on the indicated plant parts of DM. The leaf showed thehighest effects on the increment of the OL membrane size. Bars represent mean+ s.e.m. *p , 0.05; ***p , 0.001; n ¼ 3experiments, Student’s t-test. Scale bars, 100 mm (a – d,f – m).
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Figure 2. Dendropanax morbiferus leaf extracts downregulated inhibitory factors and upregulated promoting factors for OLdifferentiation in pure OPC cultures. (a – d ) OPCs were treated with EtOH- or DM leaf extract-containing medium during DIV1 – 3 and were used for RNA extraction and cDNA synthesis, followed by real-time PCR to ascertain the relative expressionpattern of the following genes: (a) Olig1, (b) ID2, (c) Ascl1 and (d ) MBP. The expression of Olig1 and ID2 but not Ascl1 wassignificantly downregulated, while the expression of MBP was in a tendency to increase by DM leaf extract treatment. Barsrepresent mean+ s.e.m. *p , 0.05; **p , 0.01; n ¼ 3 experiments, Student’s t-test. (e) Three different cultures werepooled and used for western blot analysis to detect MBP and phospho-p42/44. b-actin was used for normalization. Comparedwith EtOH-treated cultures, upregulations in MBP ( f ) and in phosphorylation of p42/44 (g) were observed in the DM leafextract-treated cultures.
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synthesis and real-time PCR. Gene expressions related to OL differentiation, such as Olig1, ID2, Ascl1 andMBP, were investigated. GAPDH was used to normalize the expression of the indicated genes. The
relative expression of Olig1 and ID2 were significantly decreased (figure 2a,b), while that of MBP
showed a tendency of increase (figure 2d ) compared with EtOH control. The relative expression of
Ascl1 was not changed by the treatment (figure 2c). During OPC differentiation, the expressions of
Olig1 [29], ID2 [30] and Ascl1 [31] are downregulated, whereas MBP expression is upregulated [32].
Therefore, real-time PCR results suggest that the DM leaf extract contains biologically effective
components for OL differentiation, supporting the immunocytochemistry data, which showed the
positive effects of DM leaf on OL membrane synthesis.
Next, to see whether we can find changes in protein levels, we performed western blot analysis in the
same system on DIV 3 using OPCs treated with either DM leaf or control during DIV 1–3. Consistent
with the real-time PCR results, MBP expression normalized by b-actin was upregulated in DM leaf
extract-treated OPC cultures compared with the control-treated OPC cultures (figure 2e,f; electronic
supplementary material, figure S3). The extracellular signal-regulated kinase (ERK) signalling pathway
plays a specific role in the timing of OL differentiation [33]. Therefore, we also checked the
phosphorylation status of ERK1/2 to see whether the altered differentiation by DM leaf extract
treatment affects ERK signalling. The results showed that the DM leaf extract treatment increased the
phosphorylation of ERK1/2 compared with the control treatment (figure 2e,g), suggesting that the
enhanced OL differentiation by DM leaf extract is mediated by ERK signalling to a certain extent.
3.5. Dendropanax morbiferus leaf extracts enhanced the oligodendrocyte differentiationin the myelinating cultures
Increases in OL membrane size (figure 1) and changes in OL gene expression (figure 2) in OPC cultures
by DM leaf extracts suggest that DM leaf extracts may enhance OL differentiation in OPC cultures.
In vivo, there are numerous neuronal signals affecting OL differentiation and myelin synthesis.
Therefore, to know whether the DM leaf extracts affect OL differentiation even under the existence of
neurons, we used in vitro myelinating cultures, in which pure OPCs are seeded on DRG neurons. To
analyse the OL differentiation step, we used DIV 7 cocultures for immunostaining of antibodies
against Olig2 (positive for all OL stages), O4 (positive from premature OL stage) and MBP (positive
from mature OL stage) (figure 3a). At this time point (DIV 7), myelin is not formed; however, OL
differentiation can be distinguished by the indicated markers of various OL developmental stages. For
in vitro cocultures, DM leaf extracts of different age were used to check whether or not they show the
same tendency in their effects on OL membrane development. The number of Olig2-positive cells per
field (figure 3c) as well as O4-positive area per cell (figure 3d ) remained unchanged. The MBP-
positive area per cell significantly increased in cocultures incubated with 6- and 20-year-old DM leaf
extracts (*p , 0.05, Student’s t-test, n ¼ 3 experiments, figure 3e) but not with 50-year-old DM leaf
extract (figure 3e). These results suggest that extract from DM leaf enhances the OL differentiation not
only in pure OPC cultures (figure 1f–o) but also in DRG/OPC cocultures (figure 3). Moreover, it
indicates that DM leaf does not have a significant effect on OPC proliferation (figure 3c).
3.6. Dendropanax morbiferus leaf extract increased the axonal contacts of oligodendrocytesin the myelinating cultures
We found that DM leaf extract enhanced OL differentiation on DIV 7 in DRG/OPC cocultures (figure 3).
Next, we asked whether the facilitated differentiation enhances axonal contacts of OLs to initiate
myelination. To test this, we immunostained cocultures on DIV 12 with antibodies against Caspr,
which clusters upon OL contact and ensheathment [34,35]. Caspr-positive area per cell was measured
and compared between EtOH- (figure 4a) and DM leaf extract-treated cultures (figure 4b). The result
showed that DM leaf extract significantly facilitated Caspr clustering in DRG/OPC coculture system
on DIV 12 (*p , 0.05, Student’s t-test, n ¼ 3 experiments, figure 4d ). This suggests that DM leaf extract
is effective not only for OL differentiation but also in boosting axonal contacts.
To confirm the immunocytochemical analysis in the myelinating culture system, we performed
quantitative real-time PCR on myelin genes by using the same culture system on DIV12. Myelin genes
such as Olig1, Myrf, CNP, MBP and PLP were investigated. Myrf is a transcription factor activating
the expression of myelin genes [36]. CNP is a non-compact myelin protein [37]; MBP and PLP are
important structural components of compact myelin [38]. We found that all of the investigated myelin
100 mm
EtOH 6-year 20-year 50-yearOlig2 O4
MBPM
BP/
O4/
Olig
2/D
api
Dap
iO
lig2
O4
MB
P
Dendropanax morbiferus leaf EtOH extract
DRG-OPC (DIV7)
EtOH
100
80
60
40
20
Olig
2+ c
ell n
umbe
r pe
r fi
eld
(580
313.
0862
mm
2 )
O4+
are
a (m
m2 )
/Olig
2+
cell
num
ber
MB
P+ a
rea
(mm
2 )/O
lig2+
cell
num
ber
0
5000
4000
3000
2000
1000
0
2500
2000
1500
1000
500
0
6-year 20-year 50-yearDendropanax morbiferus
leaf extract
EtOH 6-year 20-year 50-yearDendropanax morbiferus
leaf extract
EtOH 6-year 20-year 50-yearDendropanax morbiferus
leaf extract
**
(a) (b)
(c)
(d)
(e)
Figure 3. Dendropanax morbiferus leaf extract enhanced the OL differentiation in the late stage of OL development in themyelinating cultures. (a) For in vitro cocultures, OPCs were seeded on dorsal root ganglion neuronal cultures (DIV 0) andincubated with the medium containing control or DM leaf extract of the indicated ages. Fresh medium was added every twodays. On DIV 7, cocultures were incubated with O4-containing medium and subsequently fixed for further staining withantibodies to MBP, Olig2 and Dapi. (b) OL developmental markers used in the analysis are indicated. Antibodies to Olig2(blue), O4 (green) and MBP (red) stain OL-lineage cells from precursor, immature and mature stages, respectively. Imageanalyses were performed in the following aspects: (c) Olig2-positive cell number per field, (d ) O4-positive area per an OL-lineage cell and (e) MBP-positive area per an OL-lineage cell. There were no significant changes in the early stage of the OLdevelopment by DM leaf treatment (c,d ). However, DM leaf of 6- and 20-year-old but not 50-year-old significantly increasedMBP-positive area per cell at DIV 7 (e). Bars represent mean+ s.e.m. *p , 0.05; n ¼ 3 experiments, Student’s t-test. Scalebar, 100 mm.
royalsocietypublishing.org/journal/rsosR.Soc.open
sci.6:1902669
genes increased its expression by DM leaf treatment and significant increases were observed in Myrf,
CNP and PLP (*p , 0.05, **p , 0.01, Student’s t-test, n ¼ 3 experiments, figure 4e). The increase in
myelin gene expression is consistent with the immunocytochemical analysis (figure 4a–d ), supporting
the facilitating effects of DM leaf on myelination.
3.7. Chemical composition of Dendropanax morbiferus leaf extract by LC-MS/MS analysisTo know the chemical constituents of DM, we performed LC-MS/MS analysis by using DM leaf EtOH
extracts. A total of 300 chemicals were assigned (data not shown) and their molecular weights were
matched with databases. Among these, compounds which have been reported as their bioactivities are
listed in table 1. The lists include quercetin [39–41], chlorogenic acid [42], rutin [39,40], carnosol [43],
dextromethorphan [44], cannabidiol [45], bremazocine [46], doxapram [47], resolvin D2 [48],
procyclidine [49–51], 2-arachidonoylglycerol [52–55] and eplerenone [56].
4. DiscussionIn this paper, we report the function of DM on the development of OL-lineage cells: from OPC
proliferation till myelination. OPCs proliferate to supply OLs producing enough myelin for the
nervous system. By DM treatment, the number of OLs was not significantly changed (figure 3c),
suggesting DM does not affect the proliferation of OPCs. In the OPC stage, they express
Caspr Caspr
Cas
pr
Dendropanax m. leaf extractEtOH
oligodendrocyte
EtOH DM leaf
0 0Olig1 Myrf CNP MBP PLP
DM leafEtOH
* **
*
*
1.0
0.5
1.5
2.0
2.5
3.0
20
40
60
Cas
pr-p
ositi
ve a
rea
per
cell
(mm
2 )
rela
tive
expr
essi
onno
rmal
ized
by
GA
PD
H80
100
120
axon
(a) (b)
(c) (d) (e)
50 mm
Figure 4. Dendropanax morbiferus leaf extract increased the axonal contacts of OLs on DIV 12 in the myelinating cultures. (a – d )DRG – OPC cocultures were incubated with either EtOH (a) or DM leaf EtOH extract (b) containing medium during DIV 1 – 12 withregular medium changes every two days. On DIV 12, cocultures were fixed for Caspr staining to compare the axonal contacts. (c) Adrawing description showing axonal Caspr clustering by OL contacts. (d ) Image analysis of Caspr staining. All Caspr-positive area percell was combined and compared between EtOH- and Dendropanax morbiferus leaf extract-treated cocultures. Bars representmean+ s.e.m. *p , 0.05; n ¼ 3 experiments, Student’s t-test. Scale bar, 50 mm. (e) Quantitative real time PCR on themyelinating cultures treated with EtOH- or DM leaf extract during DIV 1 – 12. Several important myelin genes such as Myrf,CNP and PLP showed a significant increase in their expression by DM leaf extract treatment in the myelinating culture system.
royalsocietypublishing.org/journal/rsosR.Soc.open
sci.6:19026610
differentiation inhibitory transcription factors such as ID2 [57]. DM treatment reduced ID2 expression
(figure 2b), suggesting its facilitation of OL differentiation. Indeed, the expression of MYRF, a
transcription factor of myelinating OL [57], was significantly increased by DM treatment in
myelinating cultures on DIV12 (figure 4e), supporting this notion. Consistently, myelin genes such as
CNP and PLP showed a significant increase in their expression in DM-treated myelinating cultures
(figure 4e). MBP protein expression was also increased in all of the investigated culture systems by
DM treatment (figures 1, 2e and 3). This suggests that DM facilitates the OL differentiation process
from OPC to mature OLs forming myelin. The OPC differentiation is mediated by multiple
combinatory mechanisms [58]. Among them, ERK signalling is one of the critical pathways in OL
differentiation [33] as well as myelin growth [59]. Our results indicate that DM leaf extract enhances
OL differentiation at least partially by activating ERK signalling pathway (figure 2e,g).
For myelination, the processes of OL should contact axons, and then they initiate ensheathment to
form myelin by increasing their membrane size. To analyse the effects of DM on myelination steps,
we checked the axonal contacts of OLs by using myelinating cultures with Caspr staining (figure 4),
and investigated OL membrane synthesis by using glia mixed cultures, OPC cultures and myelinating
cultures with MBP staining or expression analysis (figures 1–4). For immunocytochemical membrane
synthesis analysis, we used mixed glial cultures and OPC cultures because they present membrane
sheath in a flat round form without neurons, which makes analysis easier. DM treatment increased
OL axonal contacts as well as OL membrane size compared with control treatment. This is consistent
with the result of facilitated OL differentiation by DM.
Tabl
e1.
Chem
icalc
ompo
nent
sof
leafe
than
olex
tracts
ofDM
.Phy
toch
emica
lsof
DMlea
feth
anol
extra
ctswe
rein
vesti
gate
dby
LC-M
S/M
San
alysis
.Che
mica
lsw
ithre
porte
dbi
oacti
vities
are
liste
d.
form
ula
sugg
este
dco
mpo
und
sugg
este
dstr
uctu
reM
S/M
S(d
atab
ase
sear
chsc
ore)
mol
ecul
arwe
ight
rete
ntion
time
(min
)pe
akar
ea(a
rbita
ryun
it)
C15
H10
O7qu
erce
tin89
.730
2.04
141
2.35
390
013.
4387
6
C16
H18
O9ch
loro
geni
cac
id89
.435
4.09
412
2.04
341
159
9.96
64
C27
H30
O16
rutin
87.7
610.
1515
72.
365
754
591.
1196
C20H
26O4
carn
osol
7533
0.18
224
7.53
73
896
034.
619
C18
H25
NO
dext
rom
etho
rpha
n66
.927
1.19
308
9.72
531
035
9.81
27
C21
H30
O2ca
nnab
idiol
—31
4.22
411
9.72
738
541.
9982
4
C20
H29
NO2
(2)-b
rem
azoc
ine
—31
5.21
991
8.21
132
23.9
5241
1
C24
H30
N2O2
doxa
pram
—37
8.22
9610
.403
3724
.664
755 (C
ontin
ued.
)
royalsocietypublishing.org/journal/rsosR.Soc.open
sci.6:19026611
Tabl
e1.
(Con
tinue
d.)
form
ula
sugg
este
dco
mpo
und
sugg
este
dstr
uctu
reM
S/M
S(d
atab
ase
sear
chsc
ore)
mol
ecul
arwe
ight
rete
ntion
time
(min
)pe
akar
ea(a
rbita
ryun
it)
C22
H32
O5re
solvi
nD2
—37
6.22
452
9.89
429
21.5
9215
5
C19
H29
NO
proc
yclid
ine
—28
7.22
368
1.59
569
6.06
3742
7
C23
H38
O42-
arac
hido
noylg
lycer
ol—
378.
2752
915
.155
5438
.022
477
C24
H30
O6ep
leren
one
—41
4.20
274
4.29
343
99.0
7077
6
royalsocietypublishing.org/journal/rsosR.Soc.open
sci.6:19026612
royalsocietypublishing.org/journal/rsosR.Soc.open
sci.6:19026613
Previous reports about DM are about antioxidant effects [2,3], cell-protective effects [4], selective celldeath [5,6], anti-thrombotic [7] and anti-inflammatory effects [8,9]. Our observation of DM functions in
enhancement in OL development is novel, raising possibilities of its potential use in multiple sclerosis
treatment. Non-targeted metabolome analysis by LC-high-resolution MS assigned multiple
compounds in DM ethanol extracts, showing DM is a complex of mixture of phytochemicals (table 1).
They contain molecules having bioactivities, such as quercetin, chlorogenic acid, rutin, carnosol,
dextromethorphan, cannabidiol, bremazocine, doxapram, resolvin D2, procyclidine, 2-arachidonoylglycerol
and eplerenone. Interestingly, quercetin, dextromethorphan and cannabidiol protect OL-lineage cells
from the stress environments [41,44,45], while quercetin also improves myelination in the context of
perinatal cerebral hypoxia ischaemia-induced brain injury by promoting the proliferation of OPCs [41].
Other components have been reported for various functions. Quercetin, chlorogenic acid and rutin
mediate the anti-oxidation process [39,42]. Carnosol and resolvin D2 are related to anti-inflammatory
functions [43,48]. Bremazocine is a k-opioid agonist [60] and Doxapram is a respiratory stimulant
[61]. Procyclidine is a synthetic anticholinergic agent [49] and 2-Arachidonoylglycerol is an
endocannabinoid that activates the cannabinoid receptors CB1 and CB2 [62–64]. Eplerenone has been
reported to reduce both the risk of death and the risk of hospitalization among patients with systolic
heart failure and mild symptoms [65]. As other components (except quercetin) have not been reported
in relation with myelination, they warrant further studies to screen the effective components on
myelination.
Accumulative findings suggest that treatment of progressive multiple sclerosis should be a
combinatory strategy of anti-inflammation, neuroprotection and regeneration [66]. The reported
functions of phytochemicals of DM leaf on inflammation and cell protection as well as our current
finding of DM leaf function in myelination indicate that DM leaf extract can be considered as a
candidate for relieving the symptoms of progressive multiple sclerosis, even as a complex. Medicinal
plants used in multiple sclerosis care report their benefits in relieving spasticity, pain, tremor and
depression [67] and enhancing fatty acid metabolism and lymphocyte function [68,69]. However, to
our knowledge, no study has explored the function of medicinal plants on OL differentiation/
myelination, which is a critical step for remyelination in multiple sclerosis, and our result that DM leaf
extracts affect OL development is the first observation as far as we know.
In this research, we have shown that DM enhances endogenous OL differentiation, followed by
membrane synthesis, using various types of primary cultures such as glial mixed culture, pure OPC
culture and myelinating cultures. However, in vivo is a much more complicated environment than
in vitro. Therefore, confirmation of the DM effects in in vivo demyelination models such as cuprizone
assay, lysolecithin mode, or experimental allergic encephalomyelitis may be necessary in the future. In
phytochemical analysis, we identified bioactive components whose functions in myelination have not
been reported yet, warranting further studies on the isolation of active components for OL
differentiation and myelination from the current isolated candidates.
5. ConclusionDM leaf EtOH extract enhanced the differentiation of OL and membrane formation, at least partially, by
ERK signalling pathway. Moreover, DM leaf extract increased the axonal contacts of mature OLs, which
is an initial step for myelination, as well as myelin gene expression. It also contained multiple bioactive
molecules, warranting further studies on searching for active components on myelination. Our findings
suggest that DM leaf extract may contain a novel therapeutic target in the treatment of progressive
multiple sclerosis.
Ethics. Permission to provide samples that were used in the study was granted to Mago-Yeongnong-Johap and Jirisan
Cheongjung Yakcho by Gyungsangnamdo Sancheongguncheong (permit no. 2012-Gyungnamsancheong-00005). The
study was reviewed and approved by the Institutional Animal Care and Use Committee (approval no. 2017-AE-01) of
the University of Brain Education.
Data accessibility. All data have been submitted as figures in the main text or as electronic supplementary material.
Authors’ contributions. J.-Y.K. and G.-J.S. participated in the design of the study and drafted the manuscript. J.-Y.Y. carried
out the molecular laboratory work, participated in data analysis and carried out the statistical analyses. Y.S. carried out
the mass spectrometry analysis. S.-K.L. and J.-D.P. coordinated the study. H.-J.Y. carried out the molecular laboratory
work, participated in data analysis, participated in the design of the study and drafted the manuscript. All authors
gave final approval for publication.
Competing interests. The authors declare no competing interests.
royalsocietypu14
Funding. This work was supported by the Basic Science Research Program through the National Research Foundationof Korea (NRF) funded by the Ministry of Education (2017R1D1A3B03027875); University of Brain Education (2017-03)
and a faculty research grant of Yonsei University College of Medicine for (6-2014-0045). The funder played no role in
the design and conduct of the study; collection, management, analysis and interpretation of the data; preparation,
review, or approval of the manuscript; and decision to submit the manuscript for publication.
Acknowledgements. We thank Dr Elior Peles for antibodies to O4 and Caspr.
blishing.org/
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